During the past 20 years there has been increasing recognition that Vibrio cholerae of O groups other than 1 (non-O1 V. cholerae) are a significant cause of diarrheal disease. Non-O1 V. cholerae have a worldwide distribution: they have been identified in epidemic form during cholera epidemics, have been associated with foodborne and waterborne outbreaks, and are a cause of sporadic cases of diarrhea in developing countries and in the United States. In an effort to identify factors associated with virulence, we administered three non-O1 V. cholerae strains to volunteers in doses as high as 109 CFU. Our data demonstrate that non-O1 strains are able to cause human disease, with illness in some cases matching the severity seen with V. cholerae O1. Our data also lead to the hypothesis that illness due to non-O1 V. cholerae is dependent both on production of a toxin, such as the non-O1 V. cholerae heat stable toxin (NAG-ST), and on the ability of a strain to colonize the intestine. We propose to test this hypothesis, and identify and characterize the virulence mechanisms involved. Molecular genetic techniques will be used to construct an isogenic ST-negative mutant of our virulent challenge strain, to determine if loss of ST results in loss of virulence. Oligonucleotides based on the cloned and sequenced ST gene will be used to determine the frequency with which the ST gene is present among clinical and environmental non-O1 V. cholerae isolates; probe-positive isolates will be further evaluated to determine if gene expression is consistent among strains. Colonization factors will be identified by mutagenizing our challenge strain with the transposon TnphoA and selecting for colonization deficient mutants. Probes derived from regions flanking critical transposon insertions will, in turn, be used to determine how widely distributed specific colonization-associated genes are among non-O1 V. cholerae strains. This work should provide us with an understanding of how non-O1 V. cholerae cause illness, with potential practical applications in development of control strategies for this widely distributed pathogen. By increasing our appreciation of basic bacterial virulence mechanisms, these data should also contribute to our overall understanding of the pathogenesis of diarrheal disease.